Field of the Invention
The present invention relates to stabilized protective antigen complexes and compositions, methods, and kits related to the same.
Description of Related Art
Protein subunit vaccines tend to be unstable and readily undergo physical and/or chemical degradation. There is a need for stabilized vaccines not only for storage during distribution, but for the preservation of native structures that are critical to inducing protective immunity.
The etiologic agent of anthrax (Bacillus anthracis) is a potential threat as an agent of biowarfare or bioterrorism because exposure to aerosolized B. anthracis spores can be lethal to mammals, such as humans. Anthrax toxin is a member of the class of bacterial toxins termed A-B toxins. A-B toxins are composed of two moieties. The A moiety is the enzymic portion of the toxin that catalyzes the toxic effect upon a cytoplasmic target within a target cell. The B moiety binds to a cellular receptor and facilitates the translocation of the A moiety across the cell membrane into the cytoplasm of the cell.
The B moieties of A-B toxins from tetanus, botulinum, diphtheria, and anthrax all form channels in membranes. The A and B moieties of anthrax toxin are secreted from the bacterial cell as distinct polypeptides. The A and B subunits of other A-B toxins are produced as single chain polypeptides or as separate chains that are assembled into oligomeric toxins before release from the bacteria.
The A-B toxin secreted from Bacillus anthracis is comprised of the B moiety protective antigen (“PA”), and the A moieties edema factor (“EF”), and lethal factor (“LF”). EF is a calmodulin-dependent adenylate cyclase which may protect the bacteria from destruction by phagocytes. LF is a metalloprotease that can kill macrophages or, at lower concentrations, prevent macrophages from secreting cytokines, resulting in a compromised host immunity. PA is a channel forming polypeptide that allows entry of EF and LF across membranes into the cell, a step that is critical for the pathogenesis of anthrax. PA is secreted as a four-domain, 83 kD protein that recognizes on the host cells the von-Willebrand factor A domain (“VWA”) of two integrin-like receptors: anthrax toxin receptor 1, (“ANTXR1”, formerly anthrax toxin receptor-tumor endothelial marker 8), and anthrax toxin receptor 2 (“ANTXR2”, formerly capillary morphogenesis protein 2 (“CMG2”)). Binding of PA to the receptor results in the proteolytic cleavage of PA by a furin-like protease on the cell surface, releasing the first 167 amino acid residues of domain 1. Thus, the C-terminal 63 kDa fragment (“PA63”) remains bound to the cell and the N-terminal 20 kDa fragment (“PA20”) dissociates from PA63. This proteolytic cleavage and subsequent dissociation of PA20 confer at least two new properties on PA63: (1) the ability to oligomerize into a ring-shaped heptameric sodium dodecyl sulfate (“SDS”)-dissociable structure termed prepore and (2) the ability to bind EF and LF, which bind with a stoichiometry of three per heptameric prepore. Binding of PA to the receptor also initiates receptor-mediated endocytosis into an endosomal compartment, which eventually becomes acidified. The low pH within the endosome induces a conformational change in the protective antigen that results in the formation of a membrane spanning channel, and this new conformation of the entire PA heptamer is termed the pore. The pore allows the transport of EF and LF into the cytosol. The exact pH required for pore formation is dependent upon interactions with the receptor—in vitro studies indicate that the pH is about 5 if the receptor is ANTXR2, and a slightly higher pH (about 6) if the receptor is ANTXR1. The receptor then dissociates from PA, allowing conformational changes to occur throughout the protein such that PA forms a membrane spanning pore. Modified forms of PA have been developed for use as vaccine and antitoxins, including those described in U.S. Pat. No. 7,731,979, incorporated by reference herein in its entirety. However, storage and stability of such proteins remains a significant challenge.